Glycolytic Disruption Triggers Interorgan Signaling to Nonautonomously Restrict Drosophila Larval Growth

Drosophila larval growth requires efficient conversion of dietary nutrients into biomass. Lactate Dehydrogenase (Ldh) and Glycerol-3-phosphate dehydrogenase (Gpdh1) support larval biosynthetic metabolism by maintaining NAD+/NADH redox balance and promoting glycolytic flux. Consistent with the cooperative functions of Ldh and Gpdh1, the loss of both enzymes, but neither single enzyme, induces a developmental arrest. However, Ldh and Gpdh1 exhibit complex and often mutually exclusive expression patterns, suggesting that the Gpdh1; Ldh double mutant lethal phenotype could be mediated nonautonomously. Here we find that the developmental arrest displayed by the double mutants extends beyond simple metabolic disruption and instead stems, in part, from changes in systemic growth factor signaling. Specifically, we demonstrate that this synthetic lethality is linked to the upregulation of Upd3, a cytokine involved in the Jak/Stat signaling pathway. Moreover, we demonstrate that either loss of the Upd3 or dietary administration of the steroid hormone 20-hydroxyecdysone (20E) rescue the synthetic lethal phenotype of Gpdh1; Ldh double mutants. Together, these findings demonstrate that metabolic disruptions within a single tissue can nonautonomously modulate interorgan signaling to ensure synchronous developmental growth.


ABSTRACT
Drosophila larval growth requires efficient conversion of dietary nutrients into biomass.Lactate Dehydrogenase (Ldh) and Glycerol-3-phosphate dehydrogenase (Gpdh1) support larval biosynthetic metabolism by maintaining NAD + /NADH redox balance and promoting glycolytic flux.Consistent with the cooperative functions of Ldh and Gpdh1, the loss of both enzymes, but neither single enzyme, induces a developmental arrest.However, Ldh and Gpdh1 exhibit complex and often mutually exclusive expression patterns, suggesting that the Gpdh1; Ldh double mutant lethal phenotype could be mediated nonautonomously.Here we find that the developmental arrest displayed by the double mutants extends beyond simple metabolic disruption and instead stems, in part, from changes in systemic growth factor signaling.Specifically, we demonstrate that this synthetic lethality is linked to the upregulation of Upd3, a cytokine involved in the Jak/Stat signaling pathway.Moreover, we demonstrate that either loss of the Upd3 or dietary administration of the steroid hormone 20-hydroxyecdysone (20E) rescue the synthetic lethal phenotype of Gpdh1; Ldh double mutants.Together, these findings demonstrate that metabolic disruptions within a single tissue can nonautonomously modulate interorgan signaling to ensure synchronous developmental growth.

SUMMARY STATEMENT
We used the fruit fly Drosophila melanogaster to demonstrate that disruption of glycolysis within a single larval tissue alters systemic cytokine signaling and nonautonomously inhibits development of the entire animal.

INTRODUCTION
Animal development requires the precise integration of environmental and metabolic cues with intrinsic growth signaling pathways (Miyazawa and Aulehla, 2018).
Under ideal growth conditions, the metabolic pathways involved in central carbon metabolism efficiently convert dietary nutrients into the necessary biomass and energy to support rapid growth.Conversely, environmental stressors such as starvation, infection, and toxicant exposure necessitate metabolic reprogramming to maintain survival until conditions improve for growth (Bland, 2023, Deng and Kerppola, 2013, Koyama et al., 2020).The coordination between growth and metabolism under stressful environmental conditions, however, is complex as there is a need to monitor and coordinate metabolic flux across all tissues of the developing animal (Kim et al., 2021).When a single tissue encounters metabolic limitations, peripheral tissues must adjust their growth rates to prevent asynchronous development.This topic of interorgan metabolic communication has become a burgeoning area of research, with numerous studies examining how nutrient-sensing proteins and secreted signaling molecules coordinate growth across multiple tissues (Rajan and Perrimon, 2011, Droujinine and Perrimon, 2016, Castillo-Armengol et al., 2019).Nevertheless, the specific metabolic signals that trigger interorgan growth signaling are only beginning to emerge (Baker and Rutter, 2023, Wang and Lei, 2018, Figlia et al., 2020).
The fruit fly Drosophila melanogaster is a powerful system for exploring how metabolism is coordinated with developmental growth (Gillette et al., 2021).The ~200fold increase in body mass that occurs during the four days of Drosophila larval development represents an appealing model for exploring metabolic mechanisms that convert nutrients into biomass and energy.In this regard, previous studies revealed that this impressive growth rate is supported by a coordinated increase in the expression of genes involved in glycolysis, the pentose phosphate pathway, as well as Ldh (Rechsteiner, 1970, Tennessen et al., 2011, Tennessen et al., 2014).The resulting larval metabolic program exhibits hallmark features of aerobic glycolysis -a specialized form of carbohydrate metabolism that is ideally suited for rapid biomass production.
Considering that several types of cancer cells also rely on aerobic glycolysis for growth and survival (Vander Heiden et al., 2009), Drosophila larvae present a compelling model to study this biosynthetic state.
We previously demonstrated that the larval metabolic program requires the enzymes Ldh and Gpdh1, which cooperatively regulate NAD + /NADH redox balance and promote glycolytic flux (Li et al., 2019).Intriguingly, while the loss of either single enzyme has minimal effect on overall larval growth, Gpdh1; Ldh double mutants experience a developmental arrest, suggesting a functional redundancy, where each enzyme partially compensates for the loss of the other (Li et al., 2019).However, neither Ldh nor Gpdh1 are ubiquitously expressed in larvae (Rechsteiner, 1970, Li et al., 2019, Rai et al., 2024), raising questions as to how these enzymes serve compensatory roles if not expressed in a strictly overlapping pattern.Here we address this question by examining the tissuespecific functions of both enzymes.
Our analysis of Gpdh1 and Ldh expression during larval development confirms previous observations that these enzymes are expressed in a complex and often mutually exclusive expression pattern (Rechsteiner, 1970).Moreover, we demonstrate that the loss of both enzymes within a single tissue inhibits larval growth in a nonautonomous manner.Subsequent RNA-seq analysis of Gpdh1; Ldh double mutants reveals altered expression of several secreted signaling molecules, including increased expression of the cytokine Upd3, which was previously shown to regulate systemic larval growth in response to cellular stress (Romao et al., 2021).Using a genetic approach, we find that upd3; Gpdh1; Ldh triple mutants can survive larval development and eventually eclose into adults, indicating that increased Upd3 expression serves a significant role in the synthetic lethal phenotype of Gpdh1; Ldh double mutants.Finally, we demonstrate that the steroid hormone 20E also plays a role in this phenotype, as a dietary supplement of 20E also allows Gpdh1; Ldh double mutants to complete larval development.Overall, our findings reveal that the developmental delay and lethal phenotype associated with Gpdh1; Ldh double mutants are not simply the result of metabolic failure but rather stem, in part, from changes in systemic growth signaling.

Ldh and Gpdh1 influence larval tissue growth in a nonautonomous manner
To better understand how Ldh and Gpdh1 cooperatively promote Drosophila larval growth, we examined the spatial expression pattern of both enzymes.Our analysis revealed that Ldh and Gpdh1 are often expressed in a mutually exclusive manner.In the Malpighian tubules, for example, Ldh is expressed in the stellate cells while Gpdh1 is primarily expressed in principal cells (Figure 1A-A''').Similarly, cells of the larval midgut tend to express high levels of either Ldh or Gpdh1, but rarely do we observe a cell expressing both enzymes (Figure 1B-B''', S1A-D).This trend is also apparent in the central nervous system, where although both Ldh and Gpdh1 are coexpressed in many cells the CNS, Gpdh1, but not Ldh, is expressed in neural stem cells and the prothoracic gland (Figure 1C-D''' and S1E-H).Finally, both the fat body and salivary glands express Gpdh1 but not Ldh (Figure 1A-A''', S1I-P), a result that is consistent with previous observations.In fact, the only tissue where we consistently observed uniform and overlapping expression of both enzymes was in the larval body wall muscle (Figure 1E-E''').
The complex Ldh and Gpdh1 expression patterns raise the question as to how these enzymes cooperatively regulate larval metabolism if they are not present in the same cells and tissues.One explanation is that loss of one enzyme alters the spatial expression pattern of the other; however, we find no evidence to support this hypothesis.For example, Ldh expression is nearly undetectable in wild-type salivary glands and is not increased in Gpdh1 mutant salivary glands (Figure S2A-G).Similarly, salivary gland Gpdh1 expression is not further elevated in Ldh mutants (Figure S2H-N).We also observe no increase in Ldh expression within the fat body of Gpdh1 mutants (Figure S3A-G), although we do see a slight increase in Gpdh1 expression within Ldh mutant fat bodies (Figure S3 H-N).These results are also consistent with a previous observation that Gpdh1 levels are not increased in Ldh mutant clones within the larval CNS (Li et al., 2019).
Our findings suggest that the Gpdh1; Ldh double mutant growth defects are not simply the combined result of metabolic dysfunction within individual cells, but rather stem from changes in interorgan signaling.We tested this hypothesis by using RNAi to deplete Ldh expression in the muscle (Mef2R-GAL4) and fat body (r4-GAL4) of Gpdh1 mutant larvae.With both drivers, loss of the two enzymes within a single tissue significantly reduced larval growth (Figure 2A, S4A) and slowed development as determined by time to pupation (Figure 2B, S4B).Moreover, these developmental delays were apparent in tissues not expressing the Ldh-RNAi transgene -for example, the salivary glands of age-matched Gpdh1; Mef2R-Ldh-RNAi larvae were significantly smaller than those of either the wild-type control, Gpdh1 mutant, or Mef2R-Ldh-RNAi animals (Figure 2C-F).Together, these results suggest that loss of both enzymes within a single tissue can globally influence larval growth and development.

Upd3 is required to arrest larval growth in Gpdh1; Ldh double mutants
To better understand how Ldh and Gpdh1 nonautonomously regulate larval development, we used RNA-seq to analyze gene expression in Ldh 16/17 mutants, Gpdh1 A10/B18 mutants, and Gpdh1 A10/B18 ; Ldh 16/17 double mutants relative to genetically-matched heterozygous controls (Table S1).While our approach identified several genes that were significantly changed in both single mutants (Figure 3A-B and S5), for the purpose of this analysis we focused on those genes that were only altered in double mutants (Table S2).Among the genes that were significantly downor up-regulated in Gpdh1 A10/B18 ; Ldh 16/17 double mutants but not the single mutants, we identified four secreted factors that were previously shown to systemically regulate larval growth and metabolism: peptidoglycan recognition protein SC2 (PGRP-SC2; FBgn0043575), peptidoglycan recognition protein LA, (PGRP-LA; FBgn0035975), dawdle (daw; FBgn0031461), and unpaired 3 (upd3; FBgn0053542) (Droujinine and Perrimon, 2016, Musselman et al., 2018, Romao et al., 2021).While each of these molecules is known to function in interorgan communication and could contribute towards regulating the growth of Gpdh1; Ldh double mutants, we decided to focus on the cytokine Upd3 for the remainder of this study.
Our RNA-seq analysis indicates that upd3 expression is significantly elevated in Gpdh1; Ldh double mutants.Considering that Upd3 inhibits larval development in response to both environmental and intracellular stress (Droujinine and Perrimon, 2016, Romao et al., 2021, Yang et al., 2015), increased expression of this cytokine could serve a role in the Gpdh1; Ldh developmental arrest phenotype.We tested this possibility by generating upd3 D ; Gpdh1 A10/B18 ; Ldh 16/17 triple mutants, with the hypothesis that loss of Upd3 signaling in the double mutant background would suppress the larval arrest phenotype.Indeed, triple mutants completed larval development and entered metamorphosis at a significantly higher rate than the double mutants (Figure 3C-D).Moreover, while we never observed a Gpdh1 A10/B18 ; Ldh 16/17     double mutant complete metamorphosis, up to 30% of triple mutant larvae survived to adulthood (Figure 3E-F).We would note, however, that upd3 D ; Gpdh1 A10/B18 ; Ldh 16/17     triple mutants were significantly smaller than controls, sick, and died within 2-3 days of eclosion, indicating that not all aspects of the Gpdh1 A10/B18 ; Ldh 16/17 double mutant phenotype are regulated by Upd3.
To better understand how the upd3 mutations suppress the Gpdh1 A10/B18 ; Ldh 16/17 larval arrest phenotype, we examined both double and triple mutant larvae using a semi-targeted metabolomics approach (Table S3).Our analysis revealed no significant metabolic differences between the two strains (Figure S6).Notably, the levels of both lactate and glycerol-3-phosphate were unchanged in the triple mutant as compared with double mutant larvae (Figure S6B-C), indicating that the upd3 mutation does not restore glycolytic metabolism in Gpdh1 A10/B18 ; Ldh 16/17 double mutants.
Overall, these results suggest that Gpdh1 A10/B18 ; Ldh 16/17 double mutants do not simply die from metabolic dysfunction but rather experience a larval arrest due, in part, to increased Upd3 signaling.

Ecdysone feeding is sufficient to suppress the Gpdh1; Ldh larval lethal phenotype.
To further explore the connection between Upd3 and the Gpdh1 A10/B18 ; Ldh 16/17     larval arrest phenotype, we used a 10XStat92E-GFP reporter to identify those tissues with increased JAK/STAT signaling (Yang et al., 2015, Ekas et al., 2006).Consistent with Upd3 serving a key role in limiting systemic growth, Gpdh1 A10/B18 ; Ldh 16/17 double mutants exhibited widespread 10XStat92E-GFP reporter expression when compared with either the heterozygous control strain, Ldh 16/17 single mutant, or Gpdh1 A10/B18 single mutant.In this regard, we observed increased 10XStat92E-GFP expression in the fat body (Figure Intriguingly, we also observed significantly increased 10XStat92E-GFP expression in the prothoracic gland (PG) of double mutants relative to the single mutants (Figure 4A-D), although, we would note that Gpdh1 A10/B18 single mutants displayed elevated 10XStat92E-GFP expression relative to either the Ldh 16/17 single mutant or heterozygous control strain (Figure 4B).
A previous study of tumorous wing discs demonstrated that Upd3 signaling can suppress ecdysone production in the PG and inhibit larval development (Romao et al., 2021).Thus, our observation that 10XStat92E-GFP expression is elevated within the Gpdh1 A10/B18 ; Ldh 16/17 PG raises the possibility that the double mutant larval arrest phenotype stems, in part, from decreased ecdysone signaling.Consistent with this model, we found that the addition of 20-hydroxyecdysone (20E; the active form of ecdysone) to the larval food significantly increased the pupariation rate of Gpdh1 A10/B18 ; Ldh 16/17 double mutants (Figure 4E).Overall, these results indicate that changes in 20E signaling contribute to the Gpdh1 A10/B18 ; Ldh 16/17 double mutant synthetic lethal phenotype.

Redefining the developmental functions of "Housekeeping Genes"
Our studies unexpectedly reveal that Drosophila larval development can tolerate loss of two key enzymes that regulate cytosolic NAD + /NADH balance.These findings, while surprising, are consistent with classic studies of COX5A (also known as tenured), which revealed that loss of this electron transport chain subunit induces a specific eye development phenotype (Mandal et al., 2005).The defects induced by COX5A mutations are not simply the result of defects in ATP synthesis, but rather stem from the activation of a pathway involving AMPK and p53, which eliminates Cyclin E and induces a cell cycle arrest (Mandal et al., 2005).These foundational observations were subsequently validated by both follow-up studies and a large-scale genetic screen, which demonstrated that depletion of many genes that encode electron transport chain subunits induce distinct defects in eye development (Liao et al., 2006, Pletcher et al., 2019).Our findings are built upon these earlier studies and again demonstrate that Drosophila larvae experiencing severe metabolic disruptions don't simply die due to metabolic stress, but instead arrest development through the activity of specific growth signaling pathways.Specifically, our findings suggest that loss of both Gpdh1 and Ldh activate a previously described feedforward loop, in which elevated Upd3 signaling repressed 20E signaling (Romao et al., 2021).Conceptually, our findings offer an interesting mechanistic rationale explaining how animals in the wild might cope with developmental delays as a function of exposure to severe metabolic stressors.While factors such as starvation and hypoxia inevitably induce cell autonomous responses involving the entire organism (Schito and Rey, 2018), animals also encounter environmental insults that affect individual tissues, including bacterial and parasitic infections, toxins that target specific organs, and nutrient imbalances that impinge upon cell-specific metabolic bottlenecks (Koyama et al., 2020).
Animal development has evolved to identify these tissue-specific metabolic stresses and produce signals from effected cells that are subsequently amplified and recognized by unaffected tissues, thus allowing development to be delayed in a coordinate manner.For example, Drosophila hemocytes respond to parasitic wasp infections by inducing a cell intrinsic increase in glycolytic metabolism that not only facilitates an effective immune response but also produces an adenosine signal that remodels peripheral metabolism (Bajgar et al. 2015, Bajgar andDolezal, 2018).
Our findings that Upd3 and 20E are part of a coordinated response to tissuespecific disruption of glycolytic flux provide new insights into the coordination between metabolism and developmental growth.Upd3 has emerged as a common stress-induced cytokine that communicates cell-specific information with peripheral tissues (Woodcock et al., 2015, Gera et al., 2022), and our finding reinforces a previously proposed model in which elevated Upd3 signaling induces a feedforward loop that inhibits 20E signaling and delays Drosophila development (Romao et al., 2021).In this regard, the involvement of a steroid hormone is an important feature of the regulatory mechanism, as it allows for a single tissue to indirectly influence the developmental progression of entire animal.
Moving forward, the key question emerges of how metabolic stress regulates Upd3 signaling?Both individual metabolites as well end products of cell-specific metabolic dysfunction (e.g., redox imbalance and ROS accumulation) represent potential signals that could trigger Upd3 signaling as well as related modes of interorgan communication.
Regardless of the answer, our study highlights that core metabolic enzymes are not simple products of "housekeeping genes," but rather serve dynamic cellular functions that are fully integrated into the signaling pathways that regulate multicellular growth and development.

Immunofluorescence
Larval tissues were dissected in 1X phosphate buffer saline (PBS; pH7.0) and fixed with 4% paraformaldehyde in PBS for 30 minutes at room temperature.Fixed samples were subsequently washed once with PBS and twice with 0.3% PBT (1x PBS with Triton X-100) for 10 mins per wash.
For GFP antibody staining, fixed tissues were incubated with goat serum blocking buffer (4% Goat Serum, 0.3% PBT) for one hour at RT and stained overnight at 4 ºC with primary antibody Rabbit anti-GFP diluted 1:500 (#A11122 Thermo Fisher).Samples were washed three times using 0.3% PBT and stained with secondary antibody Alexa Fluor 488 Goat anti-Rabbit diluted 1:1000 (#R37116; Thermo Fisher) for either 4 hrs at room temperature or overnight at 4 ºC.Stained tissues were washed with 0.3% PBT, immersed in DAPI (0.5µg/ml 1X PBS) for 15 mins and then mounted with a vector shield with DAPI (Vector Laboratories; H-1200-10).
Ldh protein expression was visualized using an anti-Ldh antibody (Boster bio DZ41222) as previously described (Rai et al., 2024).For larval tissues staining with anti-Gpdh1 antibody (Boster Bio DZ41223), the same protocol was used as for anti-Ldh staining, except that the anti-Gpdh1 antibody was diluted 1:100.

Mounting and imaging
All the stained tissues were mounted using VECTASHIELD containing DAPI (Vector Laboratories; H-1200-10).Multiple Z-scans of individual tissues were acquired using the Leica SP8 confocal microscope in the Light Microscopy Imaging Center at Indiana University, Bloomington.Unless noted, figure panels contain a representative Z-scan.
For presenting the Z stack in a 2D image, the Z projection tool of Fiji was used, with maximum projection.
Quantification of Gpdh1 and Ldh antibody staining was conducted by measuring the mean intensities for Gpdh1, Ldh and DAPI in the region of interest (ROI) per image, using Fiji.Gpdh1 and Ldh mean intensities were normalized to the mean DAPI intensity for the same ROI per image and plotted by using GraphPad Prism v10.1.Statistical analysis was conducted using the Mann-Whitney test.

Imaging and quantification of larval size
Third instar larvae were collected and imaged using Leica MZ 10F microscope.
The size of the larvae was measured by drawing a line from posterior to anterior end and noting down the total length by using Fiji.Data was plotted by using GraphPad Prism v10.1.Statistical analysis was conducted using the Mann-Whitney test.

Adult imaging
Third instar wandering larvae were kept on a molasses agar plate with yeast paste.Starting on D9 after egg laying, pupae were checked for eclosion, and the newly eclosed adults were collected into a BDSC food vial.Adults were imaged by using Leica DFC 450.

Percentage pupation
The pupation rate was calculated by placing 20 synchronized L1 larvae of each genotype on a molasses agar plate with yeast paste.Starting on day 4 after egg laying, plates were monitored every 24 hours for pupae.

Sample collection and extraction for Metabolomics Analysis
Mutant and control larvae were collected at the mid-L2 stage (~60 hours after egglaying at 25ºC) and extracted as previously described (Li and Tennessen, 2018).Briefly, collected larvae were placed into a prechilled 1.5 mL centrifuge tube on ice, washed with ice-cold water, and immediately frozen in liquid nitrogen.For extraction, larvae were transferred to pretared 1.4 mm bead tubes, the mass was measured using a Mettler Toledo XS105 balance, and the sample was returned to a liquid nitrogen bath prior to being stored at -80°C .Samples were subsequently homogenized in 0.8 mL of prechilled (-20 °C) 90% methanol containing 2 μg/mL succinic-d4 acid, for 30 seconds at 6.45 m/s using a bead mill homogenizer located in a 4°C temperature control room.The homogenized samples were incubated at -20 °C for 1 hour and then centrifuged at 20,000 × g for 5 min at 4°C.The resulting supernatant was sent for metabolomics analysis at the University of Colorado Anschutz Medical Campus, as previously described (Nemkov et al., 2019).

Statistical analysis of metabolite data
The metabolomics dataset was analyzed using Metaboanalyst version 6.0 (Pang et al., 2024) with data normalized to sample mass and preprocessed using log normalization and Pareto scaling.Graphs for the relative levels of individual metabolites were plotted by using GraphPad Prism v10.1.Statistical analysis for these graphs were conducted using a Mann-Whitney test.

Ecdysone feeding
Larvae were fed ecdysone in a modified molasses agar food.115 mL of molasses and 24 grams of agar were added to 750 mL of boiling water on a stirring hot plate.The temperature was then reduced to 60ºC and 5 grams of yeast was added to the media, at which point 2 mL of media was pipetted into a 35 mm petri dish.The low agar concentration resulted in a semi-solid media gained a semi-solid texture that was suitable for diffusion of a 20E stock solution.
A 30 mM stock solution of 20E (Sigma H5142) was prepared in 90% ethanol and 3.33 µL of stock solution was added to the modified molasses food, resulting in a final concentration of 50 µM (Ono, 2014;Koyama et al., 2014).Larvae of the tested genotypes were transferred to the 20E-supplemented food plates immediately after hatching and subsequently transferred to fresh 20E-supplemented plates every 24 hrs.

Figure 1 .
Figure 1.Ldh and Gpdh1 expression patterns are complex and non-strictly

Figure 2 .
Figure 2. Tissue-specific loss of Ldh and Gpdh1 induces systemic growth defects.

Figure 4 .
Figure 4. Dietary supplementation with 20E suppresses the synthetic lethal